Abstract

We have found that in addition to being potent inhibitors of
17α-hydroxylase/C17,20-lyase and/or 5α-reductase, some
of our novel androgen synthesis inhibitors also interact with the
mutated androgen receptor (AR) expressed in LNCaP prostate cancer cells
and the wild-type AR expressed in hormone-dependent prostatic
carcinomas. The effects of these compounds on the proliferation of
hormone-dependent human prostatic cancer cells were determined
in vitro and in vivo. L-2 and L-10 areΔ
4-3-one-pregnane derivatives. L-35 and L-37 areΔ
5-3β-ol-androstane derivatives, and L-36 and L-39 areΔ
4-3-one-androstane-derived compounds. L-2, L-10, and
L-36 (L-36 at low concentrations) stimulated the growth of LNCaP cells,
indicating that they were interacting agonistically with the mutated AR
expressed in LNCaP cells. L-35, L-37, and L-39 acted as LNCaP AR
antagonists. To determine whether the growth modulatory effects of our
novel compounds were specific for the mutated LNCaP AR, competitive
binding studies were performed with LNCaP cells and PC-3 cells stably
transfected with the wild-type AR (designated PC-3AR). Regardless of AR
receptor type, all of our novel compounds were effective at preventing
binding of the synthetic androgen
methyltrienolone[17α-methyl-(3H)-R1881 to both
the LNCaP AR and the wild-type AR. L-36, L-37, and L-39 (5.0μ
m) prevented binding by >90%, whereas L-35 inhibited
binding by 30%. To determine whether the compounds were acting as
agonists or antagonists, LNCaP cells and PC-3AR cells were transfected
with the pMAMneoLUC reporter gene. When
luciferase activity was induced by dihydrotestosterone, all of the
compounds were found to be potent inhibitors of transcriptional
activity, and the pattern of inhibition was similar for both receptor
types. However, L-2, L-10, and L-36 were determined to be AR agonists,
and L-35, L-37, and L-39 were wild-type AR antagonists. When tested
in vivo, L-39 was the only AR antagonist that proved to
be effective at inhibiting the growth of LNCaP prostate tumor growth.
L-39 slowed tumor growth rate in LNCaP tumors grown in male SCID mice
to the same level as orchidectomy, significantly reduced tumor weights
(P < 0.05), significantly lowered serum
levels of prostate-specific antigen (P < 0.02), and significantly lowered serum levels of testosterone
(P < 0.05). L-39 also proved to be
effective when tested against the PC-82 prostate cancer xenograft that
expresses wild-type AR. These results show that some of our compounds
initially developed to be inhibitors of androgen synthesis also
interact with the human AR and modulate the proliferation of
hormone-dependent prostatic cancer cells. Therefore, compounds such as
L-39, which have multifunctional activities, hold promise for the
treatment of androgen-dependent prostate tumors.

INTRODUCTION

Prostatic carcinoma is the most commonly diagnosed malignancy in
men in the United States and is second only to lung cancer in
cancer-related deaths
(1, 2)
. Androgens play an important
role in controlling the growth of the normal prostate gland, and in
promoting
BPH
4
and prostatic carcinoma
(3)
. The two most important
androgens in prostate cancer etiology are testosterone and DHT.
Testosterone is synthesized primarily in the testes and also, to a
lesser extent, in the adrenals. Testosterone is further converted to
the more potent androgen DHT by the enzyme 5α-reductase, which is
localized primarily in the prostate
(4)
. Although
testosterone and DHT both stimulate the growth of normal and malignant
prostate tissue, DHT is believed to be the more important androgen
(5, 6)
.

Androgen ablation therapy has been shown to produce the most beneficial
responses in patients with hormone-responsive prostatic tumors.
Orchidectomy (castration, either surgical or medical with a luteinizing
hormone-releasing hormone analogue) remains the standard treatment
option for most patients. However, both methods, which result in
reduced androgen production by the testes, fail to alter androgen
production by the adrenal glands. Studies in the United States and
Europe have reported that although androgen ablation therapy alone is
an effective treatment, a combination therapy of orchidectomy with
antiandrogens, to inhibit the action of adrenal androgens,
significantly prolongs the survival of prostate cancer patients
(7,
8,
9)
. Given that efforts to block the production or
effects of adrenal androgens result in worthwhile therapeutic gains,
this laboratory has been designing and evaluating novel compounds that
inhibit androgen production from all sites in the body. The compounds
developed are steroidal antagonists of the steroidogenic enzymes
C17,20-lyase and 5α-reductase.
C17,20-lyase catalyzes both the
17α-hydroxylation and the cleavage of the
C17,20-side chain during the conversion of the
21-carbon steroids pregnenolone and progesterone to the 19-carbon
androgens dehydroepiandrosterone and androstenedione, respectively
(10)
. The enzyme has an identical amino acid sequence in
both testicular and adrenal tissue
(11)
, indicating that
inhibitors of this enzyme would be equally effective at both sites. Two
isoforms of 5α-reductase occur in the body (type I and type II), and
the type II enzyme is the predominant form in the human prostate.
Inhibitors of this isoenzyme would essentially prevent prostatic
accumulation of the potent androgen DHT.

A number of C17,20-lyase-inhibitors have been
described. However, most are not specific, and only the imidazole
antifungal agent ketoconazole has been used clinically to reduce
testosterone levels in patients with advanced prostate cancer
(12,
13,
14)
. The major drawback with ketoconazole is that it
is not very potent or specific. It is only a moderate inhibitor of
C17,20-lyase, inhibits cortisol production, and
has a number of significant side effects. Nevertheless, recent studies
have reported that ketoconazole was effective in reducing PSA levels in
55%
(15)
and 62.5%
(16)
of patients who had
progressed after antiandrogen (flutamide) withdrawal. These results
indicate that compounds more specific and selective than ketoconazole
may be more effective for the treatment of prostate cancer. Several
inhibitors of 5α-reductase have also been described and finasteride,
which is a more potent inhibitor of the type II than the type I
isoenzyme
(17)
, has been approved for the treatment of BPH
(18)
. Although effective at reducing DHT levels in
patients with prostate cancer, finasteride also increases the
bioavailable levels of testosterone
(19)
, which can
stimulate tumor growth
(20)
. Therefore, compounds designed
to inhibit both C17,20-lyase and 5α-reductase
would be expected to be more clinically beneficial to prostate cancer
patients than compounds which inhibit only one of the enzymes.

The growth effects of testosterone and DHT on prostate cancer cells are
mediated by the androgens binding to their cognitive nuclear receptors,
which in turn bind to specific response elements in the promoter
regions of androgen-regulated genes. The products of these genes
modulate cellular proliferation
(21)
. Antiandrogens such
as flutamide have also been used clinically for the treatment of
prostate cancer. However, the results were disappointing. Some patients
tended to improve after flutamide was withdrawn after relapse
(22, 23)
. Nonetheless, as described above, clinical trials
which combined the therapies of orchidectomy with the antiandrogen
flutamide reported a significantly longer survival period than
orchidectomy alone
(7,
8,
9)
. This implies that a treatment
regimen involving total androgen ablation in combination with an
antiandrogen may be a more effective treatment option for patients with
androgen-dependent prostate tumors.

Previously we have reported the synthesis and testing of several
steroidal inhibitors of C17,20-lyase and
5α-reductase
(24,
25,
26,
27,
28)
. These compounds were shown to be
effective inhibitors of human testicular
C17,20-lyase and prostatic 5α-reductase
in vitro (See Table 1
⇓
). In the present study we report that some of these compounds
are also very potent antiandrogens, and that this property contributes,
at least in part, to their growth-inhibitory effects on
androgen-dependent LNCaP prostate cancer cells in vitro and
in vivo. LNCaP cells are the most frequently studied
AR-positive prostate cancer cell line that can be readily grown in
tissue culture
(29,
30,
31)
. LNCaP cells are not only
androgen-responsive but also androgen-dependent. They respond to
androgens with increased cellular proliferation and elevated expression
of PSA
(32)
. Although LNCaP cells have a mutated AR
(33, 34)
, they have been used extensively for research on
the causes, treatment, and prevention of prostate cancer
(35)
. We have used these cells in culture and as tumors in
nude mice to compare the efficacy of our compounds with the known
inhibitors of C17,20-lyase and 5α-reductase,
ketoconazole, and finasteride, respectively. The antiandrogenic
properties of the compounds were compared with flutamide.

Cell Culture.

LNCaP, PC-3, and CV-1 cells were grown in RPMI 1640 medium
supplemented with 10% FBS and 1% penicillin/streptomycin solution. To
determine the effect of steroids and novel compounds on cell
proliferation, hormone-dependent LNCaP and hormone-independent PC-3
cells were transferred into steroid-free medium 3 days prior to the
start of experiments. Steroid-free medium consisted of phenol red-free
IMEM supplemented with 5% dextran-coated, charcoal-treated serum, and
1% penicillin/streptomycin solution. Serum was depleted of steroids as
described previously
(38)
. Growth studies were then
performed by plating cells (1.5 × 10
4
) in 24-place multiwell dishes (Corning, Inc.,
Corning, NY). After a 24-h attachment period, the medium was aspirated
and replaced with steroid-free medium containing vehicle or the
indicated concentrations of androgens and novel compounds. This medium
was changed every 3 days and the cells were counted 9 days later using
a Coulter Counter model Z-1 (Coulter Electronics, Hialeah, FL). Cell
numbers were expressed as a percentage of vehicle-treated cells.

Wild-type LNCaP cells and CV-1 cells were transfected with the
pMAMneoLUC plasmid as we have described previously
(39)
. Briefly, 2 × 105 cells in a 35-mm2 dish
were exposed to 3 ml of Opti-MEM containing Lipofectamine (30 μl) and
6 μg of the pMAMneoLUC plasmid for 5 h at 37°C in a
5% CO2 incubator. The medium was then changed to
routine culture medium for 72 h. Cells were then grown in medium
supplemented with 750 μg/ml G418. The surviving colonies were picked
and grown in selective media. Stable selectants were tested twice per
month for luciferase activity, as described in the later section. After
3 months (∼23 passages), the transfectants with the highest
luciferase activity were selected and designated LNCaP-LUC and CV-1LUC,
respectively. These cell lines and the PC-3AR cell line were routinely
cultured in RPMI 1640 medium supplemented with 10% FBS, 1%
penicillin/streptomycin solution, and 750 μg/ml G418.

Competitive Binding of [3H]R1881 to the LNCaP AR
and Wild-Type AR in the Presence of Novel Compounds.

Competitive binding studies with the synthetic androgen R1881
were performed essentially as described by Wong et al.(40)
and Yarbrough et al.(41)
.
Wells in 24-place multiwell dishes were coated with
poly-l-lysine (0.05 mg/ml) for 30 min and dried.
To determine the kinetics of R1881-binding to the LNCaP AR and
the wild-type AR, LNCaP cells and PC-3AR cells (2–3 × 105) were plated in steroid-free medium and
allowed to attach. The following day, the medium was replaced with
serum-free, steroid-free IMEM supplemented with 0.1% BSA and
containing [3H]R1881 (0.01–10
nm) in the presence or absence of a 200-fold
excess of cold R1881 to determine nonspecific binding and 1μ
m triamcinolone acetonitride to saturate
progesterone and glucocorticoid receptors. Following a 2-h incubation
period at 37°C, cells were washed twice with ice-cold DPBS and
solubilized in DPBS containing 0.5% SDS and 20% glycerol. Extracts
were removed and the cell-associated radioactivity counted in a
scintillation counter. The data were analyzed using RADLIG 40 software
(Biosoft, Ferguson, MO), and Kd and
Bmax determined by Scatchard plot
transformation. When the concentration of R1881 required to
almost saturate AR in both cell lines was established, the ability of
the test compounds (5.0 μm) to displace[
3H]R1881 (5.0 nm) from
the receptors was determined as described above.

Transient Transfection of PC-3AR Cells with the
pMAMneoLUC Plasmid and CV-1LUC Cells with the pCMV5-hAR
and pCMV5-LNCaPAR Plasmids.

Cells were grown in steroid-free IMEM for 3 days and plated
(4 × 10
4
cells/well) in 24-well
plates in phenol red-free IMEM supplemented with 10% charcoal-stripped
serum and no antibiotics. After a 24-h incubation period, the cells
were washed twice with DPBS and each well was incubated with 250 μl
of phenol red-free IMEM containing 2 μl of PLUS-reagent, 4 μl of
Lipofectamine, and 4 μg of the pMAMneoLUC plasmid for
PC-3AR cells and 0.5 μg of the pCMV5-hAR or pCMV5-LNCaPAR plasmids
for CV-1LUC cells. After a 5-h incubation period, 250 μl of routine
medium were added to each well and the cells were incubated for an
additional 24 h. The resultant cells, designated PC-3AR/LUC,
CV-1LUC/hAR, or CV-1LUC/LNCaPAR, were assayed for luciferase activity
as described in the following section.

Luciferase Activity Assay.

LNCaP-LUC cells were transferred to steroid-free medium 3 days
before the start of the experiment and plated at 1 × 105 cells/well in steroid-free medium. PC-3AR/LUC cells,
CV-1LUC/hAR cells, and CV-1LUC/LNCaPAR cells were transiently
transfected as described above. After a 24-h incubation period in
steroid-free medium, each well was treated with ethanol vehicle or the
selected steroids and novel compounds (5 μm) in
triplicate. After a 24-h treatment period, the cells were washed twice
with ice-cold DPBS and assayed using the Luciferase kit according to
the manufacturer’s protocol. Briefly, the cells were lysed with 200μ
l of luciferase lysing buffer, collected in a microcentrifuge tube
and pelleted by centrifugation. Supernatants (100 μl aliquots) were
transferred to the corresponding wells of white 96-well plates
(Polyfiltronics, Inc., Boston, MA). Luciferin (50 μl) was
added to each well, and the light produced during the luciferase
reaction was measured in a Victor 1420 Multilabel counter (Wallac,
Inc., Gaithersburg, MD). The effects of the steroids and novel
compounds on DHT-induced luciferase transcription were determined using
the same protocol.

In Vivo Studies with LNCaP Tumors in SCID Mice and
PC-82 Xenografts in Athymic Nude Mice.

Male SCID mice and athymic nude mice 4–6 weeks of age were purchased
from the National Cancer Institute (Frederick, MD). Animals were housed
in a pathogen-free environment under controlled conditions of light and
humidity and received food and water ad libitum.

Inoculation of LNCaP Cells into Male SCID Mice.

LNCaP tumors were grown s.c. in male SCID mice essentially as described
by Sato et al.(42)
with modifications based on
the breast cancer model described by Yue et al.(43, 44)
. LNCaP cells were grown in routine culture medium (RPMI 1640
medium supplemented with 10% FBS and 1% penicillin/streptomycin)
until 80% confluent. Cells were scraped into DPBS, collected by
centrifugation, and resuspended in Matrigel (10 mg/ml) at 3 × 107 cells/ml. Each mouse received s.c.
injections at one site on each flank with 100 μl of cell suspension.
Tumors were measured weekly with calipers, and tumor volumes were
calculated by the formula 0.5236 × r12 × r2
(r1 < r2).

Inoculation of PC-82 Xenograft Tumors Into Male Athymic Nude
Mice.

Hormone-dependent PC-82 tumor xenografts were kindly provided by Dr.
John Isaacs (John Hopkins School of Medicine, Baltimore, MD). PC-82
tumors were minced into fine pieces, washed with DPBS, filtered, and
resuspended in Matrigel at a concentration of 100 mg/ml The mice were
inoculated s.c. at one site on each flank with 100 μl of tumor
suspension using a 18-gauge needle. Tumors were allowed to grow (∼3
months) and then were measured weekly. Tumor volumes were then
calculated weekly according to the formula 0.5236 × r12 × r2 (r1 < r2). When tumors reached a measurable
size, the mice were randomized into treatment groups (five
animals/group) and treated as described in the following section.

Treatment.

For LNCaP tumor experiments, treatments began 4–5 weeks after cell
inoculation when measurable tumor volume was 500
mm3 (For PC-82 xenografts, this was ∼3 months).
For each experiment, groups of six mice (five mice for PC-82
xenografts) with comparable total tumor volumes were either castrated
or treated with the novel compounds at 50 mg/kg/day. Mice were
castrated under methoxyfluorane anesthesia via the abdominal approach.
Compounds and reference drugs were prepared at 10 mg/ml in a 0.3%
solution of hydroxypropyl cellulose in saline, and mice received s.c.
injections daily. Control and castrated mice were treated with vehicle
only. Treatments lasted for 28 days, after which time the animals were
sacrificed by decapitation and the blood was collected. Tumors were
excised, weighed, and stored in liquid nitrogen for additional
analysis.

Testosterone RIA Assays.

For measurement of serum testosterone levels, 50 μl of mouse serum
were assayed according to the instructions provided with the
125I-testosterone RIA kit supplied by DSL, Inc.
Radioactivity was counted using a Packard Cobra II gamma counter. For
measurement of tumor testosterone levels, whole tumors were homogenized
in phosphate buffer (pH 7.4; 0.1 m). The homogenates were
then centrifuged at 2000 × g for 20 min to
remove debris. Fifty-μl aliquots of the tissue supernatant were used
to determine the tumor testosterone concentration, as described above.

Measurement of Serum PSA Levels.

Serum PSA levels were determined using a PSA ELISA kit supplied by DSL,
Inc. Briefly, 2.5 μl of serum diluted 1:10 in DPBS was mixed with
assay buffer to a final volume of 75 μl and added to duplicate wells
in the 96-well plate that had been coated with an anti-PSA antibody.
Following a 1-h assay and extensive washing of the plate, the wells
were treated for 30 min with a second anti-PSA antibody labeled with
horseradish peroxidase. After washing, the wells were treated with the
tetramethylbenzidine substrate for 10 min, and the absorbance was read
at 450 nm with a Dynatech MRX plate reader.

Statistical Analysis.

One-way ANOVA on SigmaStat for Windows version 1.0 was used to compare
the different treatment groups at the 95% confidence level. A
P of <0.05 was considered to be statistically significant.

RESULTS

Growth Effects on LNCaP and PC-3 Cell Cultures in
Vitro.

The structures of the compounds are shown in Fig. 1
⇓
, and their in vitro activities against
C17,20-lyase and 5α-reductase are provided in
Table 1
⇓
. For growth studies, androgen-responsive LNCaP cells and
androgen-independent PC-3 cells were deprived of steroids for 3 days
before the experiments. After plating and attachment, cells were
treated with the compounds at concentrations of 0.1μ
m, 1.0 μm, and 5.0μ
m for 9 days (Fig. 2A⇓
and 2B)
⇓
. In agreement with our previous findings
(45, 46)
, DHT and testosterone (both at 1 nm)
were potent stimulators of LNCaP cell growth in vitro, and
increased cell number by 5.9-fold and 4.4-fold, respectively (Fig. 2A)
⇓
. Neither testosterone nor DHT had any effect on the
growth of hormone independent PC-3 prostate cancer cells (Fig. 2B)
⇓
. At each of the concentrations tested, the antiandrogen
flutamide also increased the growth rate of LNCaP cells, whereas
ketoconazole had no effect on cellular proliferation. Finasteride (1μ
m and 5 μm) inhibited
growth by 60% compared with vehicle-treated cells. L-37 was the
most potent inhibitor of cell growth and did so in a dose-dependent
manner. Names of the “L” compounds are shown in Fig. 1
⇓
. L-37
inhibited cell growth by 34% at 0.1 μm, 54%
at 1.0 μm, and 67% at 5μ
m. L-35 inhibited cell growth by 40% at both
1-μm and 5-μm
concentrations. L-10 increased cell number by approximately 2-fold at
each of the concentrations tested. L-2 was found to be the most potent
stimulator of cell growth and 0.1 μm, 1.0μ
m, and 5.0 μm L-2
increased cell number by 4.6-fold, 4.4-fold, and 2.9-fold,
respectively. L-36 showed a biphasic effect on cell growth. The lower
concentrations (0.1 μm and 1.0μ
m) increased the growth rate of LNCaP cells by
2.3-fold and 2-fold, respectively. However, at a
5-μm concentration, L-36 inhibited cell growth
by 50%. L-39 was mildly growth-stimulatory to LNCaP cells at the two
lower concentrations, but at the higher concentration of 5.0μ
m, L-39 had little effect on the growth rate.
When the compounds were tested against androgen-independent PC-3 cells
over the same 9-day treatment period, the only compounds that modulated
growth were L-37 and L-39, which at the 5.0-μm
concentration inhibited the growth of PC-3 cells by approximately 30%
(Fig. 2B)
⇓
.

The effect of the novel compounds and reference drugs on
the growth of (A) hormone-dependent LNCaP prostate
cancer cells and (B) hormone-independent PC-3 prostate
cancer cells in steroid-free medium. Cells were grown in steroid-free
medium for 3 days before plating. Triplicate wells were then treated
with 0.1 μm, 1.0 μm, and 5.0μ
m of the reference drugs and compounds for 9 days, as
described in “Materials and Methods.” Cell numbers are expressed as
a percentage of the mean number of cells in the control
(vehicle-treated) wells. The results show the mean and SE from three
separate experiments.

To determine whether the mechanism of action of the compounds was
mediated by the AR, the ability of the compounds to compete with DHT
and modulate cell proliferation was investigated (Fig. 3A)
⇓
. Flutamide and ketoconazole had no effect on DHT-induced
LNCaP cell proliferation. Surprisingly, the 5α-reductase
inhibitor finasteride reversed the growth stimulatory effect of DHT on
LNCaP cells in a dose-dependent manner. L-2 and L-10 had no effect on
DHT-induced LNCaP cell proliferation at concentrations of 0.1μ
m and 1.0 μm. At a
concentration of 5.0 μm, both compounds
slightly reduced proliferation to levels similar to those when the
compounds were tested in the absence of DHT. No synergistic or additive
effects were observed when LNCaP cells were cotreated with the growth
stimulatory compounds L-2 or L-10 and DHT. L-36 had no effect on
DHT-induced LNCaP cell proliferation at concentrations of 0.1μ
m and 1.0 μm. However
at 5.0 μm concentration, L-36 inhibited cell
proliferation by >90%. L-37 was shown to be the most potent inhibitor
of DHT-induced LNCaP cell proliferation. L-37 inhibited DHTinduced
growth by 73%, 88%, and 95% at concentrations of 0.1μ
m, 1.0 μm, and
5.0 μm, respectively. L-35 and L-39 were also
effective at reversing the growth effects of DHT and did so in a
dose-dependent manner. At the 5.0 μm
concentration, L-35 and L-39 inhibited DHT-induced LNCaP cell
proliferation by 66% and 79%, respectively. These results suggest
that the compounds may be acting to block the action of DHT in
stimulating cell proliferation.

The effect of novel compounds and reference drugs on LNCaP
prostate cancer cell growth stimulated by (A) 1
nm DHT and (B) 1 nm
testosterone. Cells were grown in steroid-free medium before plating.
Triplicate wells were then cotreated with 0.1 μm, 1.0μ
m, and 5.0 μm of the reference drugs and
compounds and the androgens, as described in “Materials and
Methods.” Cell numbers are expressed as a percentage of the mean
number of cells in the androgen-treated wells. The results show the
mean and SE from three separate experiments.

The ability of the novel compounds to compete with testosterone for the
AR and modulate cell growth in vitro was also investigated
(Fig. 3B)
⇓
. When cells were cotreated with testosterone (1
nm) and the reference drugs or novel compounds
for 9 days, the results were similar to those obtained when cell growth
was stimulated by DHT. L-37, L-35, and L-39 again proved to be the most
effective compounds at inhibiting testosterone-induced growth of LNCaP
cells in vitro. Each of these compounds inhibited growth in
a dose-dependent manner.

LNCaP and PC-3AR Androgen Receptor Binding Assays.

The ability of the novel compounds to modulate the growth of LNCaP
cells and to inhibit DHT and testosterone-induced cell proliferation
implied that they may be interacting with the LNCaP AR. To determine
whether this was specific for the mutated AR expressed in LNCaP cells,
kinetic studies were performed with the synthetic androgen
methyltrienolone [3H]R1881 binding to the
mutated AR expressed in LNCaP cells and the wild-type AR expressed in
PC-3AR cells. The data were linearized by Scatchard transformation
(data not shown) and the Kd and
Bmax for both types of AR were determined.
LNCaP cells express a single class of high affinity binding sites with
Kd = 0.78 ± 0.01
nm and Bmax = 2.60 × 105 ± 2.27 × 10
4
receptors/cell. PC-3AR
cells also express a single class of high-affinity binding sites with
Kd = 0.20 ± 0.01
nm and Bmax = 4.8 × 10
4
± 6.5 × 103 receptors/cell. The
ability of the compounds to compete with 5 nm[
3H]R1881 for binding to both types of AR was
determined (Fig. 4)
⇓
. Ketoconazole was ineffective at preventing[
3H]R1881 from binding to the AR in either cell
line. The antiandrogen flutamide prevented 52% of the[
3H]R1881 from binding to the LNCaP AR, and
51% of the labeled androgen from binding to the wild-type AR in PC-3AR
cells. Interestingly, L-35, which was one of the most effective
compounds at inhibiting the growth of LNCaP cells in vitro,
was the least effective compound at preventing[
3H]R1881from binding to either type of AR. In
LNCaP cell cultures, L-35 prevented 34% of the[
3H]R1881 from binding to the AR, and in PC-3AR
cells, the inhibition was 31%. Each of the other 5 novel compounds
were very effective at preventing [3H]R1881
from binding to the cellular AR. The pattern was similar regardless of
whether they were tested against the mutated LNCaP AR or the wild-type
AR. Moreover, they prevented [3H]R1881from
binding to the ARs with higher efficiencies than flutamide. In the
presence of L-2, L-10, L-36, L-37, and L-39, the amount of[
3H]R1881 bound to the LNCaP cells was 30%,
5.2%, 0.7%, 15.2%, and 3.3%, respectively. In PC-3AR cells, the
amount of [3H]R1881 bound in the presence of
L-2, L-10, L-36, L-37, and L-39 was 13.5%, 2.6%, 5.1%, 11.7%, and
2.5%, respectively.

Competitive binding of the synthetic androgen[
3H]R1881 to the LNCaP AR, and the wild-type AR expressed
in PC-3AR cells in the presence of the novel compounds and reference
drugs. LNCaP and PC-3AR cells were incubated with 5 nm[
3H]R1881 and 5 μm of the compounds and
reference drugs for 2 h at 37°C in serum-free medium, as
described in “Materials and Methods.” After washing and lysing,
cell-associated radioactivity was determined by liquid scintillation
counting. The amount of [3H]R1881 bound to the cells in
the presence of compound or reference drug, is expressed as a
percentage of the amount of [3H]R1881 that bound in the
presence of vehicle. The results show the mean and SE from three
separate experiments. ∗, P < 0.001
versus control; ∗∗, P < 0.01 versus control; Φ,
P < 0.05 versus
control.

To determine whether the compounds are acting as AR agonists or
antagonists, luciferase activity assays were performed using LNCaP-LUC
and PC-3AR/LUC cells. DHT (5.0 nm) significantly
(P < 0 0.001) stimulated luciferase
production mediated by the LNCaP AR in LNCaP-LUC cells (Fig. 5A)
⇓
. Ketoconazole (5.0 μm) and
flutamide (5.0 μm) also activated transcription
of the luciferase gene in LNCaP-LUC cells by approximately 2-fold each.
Finasteride had no effect on luciferase activity. L-2, L-10, and L-36
(all at 5 μm) stimulated luciferase production
in LNCaP-LUC cells by 6-fold, 4.5-fold, and 8-fold, respectively,
confirming that each of these compounds are agonists of the mutated
LNCaP AR. L-39 also, surprisingly, stimulated luciferase activity in
LNCaP-LUC cells by 2.5-fold, which suggested that it also has some
agonistic properties. L-35 and L-37 (5 μm)
significantly (P < 0.001) lowered luciferase
transcriptional activity in LNCaP-LUC cells to barely undetectable
levels, indicating that they are potent antagonists of the LNCaP AR.
These studies were also performed using PC-3AR/LUC cells, which express
wild-type AR (Fig. 5B)
⇓
. In these cells, DHT (5
nm) significantly stimulated
(P < 0.01) transcription of the luciferase
gene by 3.3-fold. This was considerably less than with LNCaP-LUC cells,
and was attributed to the cells reacting negatively to the double
transfection of foreign genes. Neither ketoconazole nor flutamide had
any appreciable effects on luciferase activity levels mediated by the
wild-type AR, suggesting that they are only agonistic with the mutated
AR. Finasteride (5 μm) did not modulate
wild-type AR-mediated luciferase activity levels. When the novel
compounds were tested, L-2, L-10, and L-36 (all at 5μ
m) significantly stimulated
(P < 0.05) luciferase production in
PC-3AR/LUC cells by 2.1-fold, 2.8-fold, and 1.7-fold, respectively,
confirming that each of these compounds are also agonists of the
wild-type AR. L-35 and L-37 (5 μm)
significantly lowered (P < 0.05) luciferase
transcriptional activity in PC-3AR/LUC cells, again indicating that
these compounds are antagonists of both the wild-type and mutant AR. In
contrast to the results obtained with LNCaP-LUC cells, L-39 inhibited
wild-type AR-mediated luciferase transcriptional activity in PC-3AR/LUC
cells by 35%, suggesting that this compound is an antagonist of the
wild-type AR and a weak agonist of the mutated LNCaP AR. The results
indicate that L-2, L-10, and L-36 function as agonists of both receptor
types, L-35 and L-37 are antagonists of both receptor types, and L-39
has mixed agonist/antagonistic activity depending on receptor type.

The effects of the novel compounds and reference drugs on
transcriptional activity of luciferase mediated through
(A) the LNCaP AR in LNCaP-LUC cells and
(B) the wild-type AR expressed in PC-3AR/LUC prostate
cancer cells. Cells in steroid-free medium were treated with vehicle,
DHT (5 nm), testosterone (5 nm), and the
reference drugs or novel compounds (5 μm) for 24 h.
Cells were then assayed for luciferase activity, as described in“
Materials and Methods.” The columns represent the mean of light
units [counts per second (cps)/unit protein] in triplicate wells from
three separate experiments. Values are expressed as mean and SE.
A, ∗P < 0.001
versus control; ∗∗P < 0.01. B, ∗P < 0.01; ∗∗P < 0.05
versus control.

The ability of the compounds to modulate DHT-induced luciferase
activity was determined in both cell types. In each of the experiments
the cells were cotreated with DHT (5 nm) and the reference
drug or novel compound (5 μm). DHT (5.0 nm)
significantly (P < 0 0.001) stimulated
luciferase production mediated by the LNCaP AR in LNCaP-LUC cells
(Fig. 6A)
⇓
. Ketoconazole had no effect on DHT-induced luciferase
activity. In this cell line, flutamide and finasteride significantly
lowered DHT-induced transcriptional activity suggesting that they
compete for the LNCaP AR and prevent DHT from binding. All of the novel
compounds significantly inhibited DHT-induced LNCaP AR-mediated
luciferase transcription. The order of potency of the compounds was
L-37 > L-39 > L-2 > L-35 > L-10 > L-36, and DHT-induced
luciferase activity was inhibited by 99.1%, 86.8%, 73.8%, 72.5%,
69.8%, and 42.1%, respectively. No synergistic or additive effects
were observed on luciferase activity levels when LNCaP-LUC cells were
cotreated with the agonistic compounds, L-2, L-10, or L-36 in
combination with DHT. These compounds compete with DHT for binding to
the mutated LNCaP AR and mediate transcription to the same level as in
the absence of DHT. When the compounds were tested against the
wild-type receptor in PC-3AR/LUC cells, a similar pattern was obtained
(Fig. 6B)
⇓
. Ketoconazole and finasteride had no effect on
DHT-induced transcriptional activity and flutamide significantly
inhibited (P < 0.01) luciferase activity
induced by DHT. As was observed with the mutated LNCaP AR, all of the
novel compounds were effective at inhibiting DHT-induced
transcriptional activity mediated by the wild-type AR. The order
of potency of the compounds was L-37 > L-39 > L-35 > L-2 ≥ L-36 ≥ L-10, which inhibited luciferase activity
levels by 89.1%, 82.2%, 69.8%, 62.9%, 59.5%, and 58.9%,
respectively.

To ensure that the results obtained with these cell lines reflected the
ability of the compounds to bind to the AR and not another steroid
hormone receptor, these assays were performed in steroid
receptor-negative CV-1LUC cells that were transiently transfected with
either the wild-type AR or the mutated LNCaP AR constructs. The results
obtained were in agreement with the results described above, indicating
that the effects of the compounds were specific for the AR (data not
shown).

Growth Effects on LNCaP Tumors Grown in Male SCID Mice.

In the first experiment, the effects of L-2, L-35, and L-36 on tumor
growth were determined, and orchidectomy and ketoconazole were used as
the reference treatments. The mice were grouped 28 days after cell
inoculation when measurable tumor volumes were approximately 500
mm3 (Fig. 7A)
⇓
. After 21 days of treatment tumor volumes in the mice
treated with L-2, L-36, and L-35 were similar to the castration group.
However, by 28 days tumor volume in the control mice increased 4-fold
over the 28 days of treatment, and tumor volume in the castrated mice
increased by only 2-fold (52% reduction). None of the treatments,
including ketoconazole, had any appreciable effect on the growth of the
tumors after 28 days of treatment (Fig. 7A)
⇓
. The reductions
in tumor volumes in the mice treated with ketoconazole, L-2, L-35, and
L-36 were 23%, 28%, 8%, and 12% respectively. These results were
also reflected in the weights of the tumors. The only treatment that
resulted in significantly (P < 0.01) smaller
tumors was orchidectomy (Fig. 7B)
⇓
. Tumor weight in the
castrated mice was reduced by 62%. Mice that had been castrated also
had significantly (P < 0.02) lower serum
levels of PSA when compared with vehicle-treated animals (Table 2)
⇓
. In this experiment, serum and tumor levels of testosterone were
significantly reduced in the mice that were castrated. Although, serum
testosterone levels in the mice treated with L-2, L-35, and L-36 were
significantly lower compared with the vehicle-treated mice, tumor
testosterone levels were unaffected.

The effects of orchidectomy, ketoconazole, and the novel
compounds L-2, L-35, and L-36 on the growth of LNCaP prostate
tumors in male SCID mice. Groups of six mice with LNCaP tumors were
treated with the compounds at 50 mg/kg/day for 28 days. A,
tumor volumes were measured weekly, and the percentage of change in
tumor volume was determined. B, after 28 days of treatment,
the mice were sacrificed and the tumors were removed and weighed.
Castration significantly reduced tumor weights. ∗,
P < 0.01 versus control.

Serum levels of PSA and testosterone and tumor levels of testosterone
in male SCID mice after 28 days of treatment with novel compounds

In the second experiment, the effects of L-10, L-37, and L-39 on tumor
growth were determined and orchidectomy, finasteride, and ketoconazole
were used as the reference treatments (Fig. 8A)
⇓
. The mice were grouped 35 days after cell inoculation when
measurable tumor volume was approximately 500 mm3
(Fig. 8A)
⇓
. Total tumor volume in the control mice increased
4.3-fold over the 28 days of treatment and tumor volume in the
castrated mice increased by only 2.6-fold (38% reduction compared with
control). Tumor volumes increased 3.2-fold in the mice treated with
finasteride and, as in the previous experiment, ketoconazole had no
appreciable effect on tumor growth. Tumor volumes in the mice treated
with L-10 increased by 3.3-fold (22% reduction compared with control)
over the 28 days of treatment. This compound was as effective as
finasteride in this model. In contrast to the growth inhibition
observed with L-37 in vitro (Fig. 2
⇓
3
⇓
4
⇓
5
⇓
6)
⇓
, tumor volumes in
the mice treated with this compound increased 5.2-fold. In the mice
treated with L-39, tumor volume increased by 2.4-fold, which was a 44%
reduction versus control mice. In this experiment, L-39 was
as effective as castration at inhibiting tumor growth. Tumor weights in
the castrated and L-39-treated mice were significantly
(P < 0.05) lower than those in the control
mice (Fig. 8B)
⇓
. Compared with vehicle-treated mice, tumors
in the castrated and L-39-treated mice weighed 75% and 72% less,
respectively. As shown in Table 3
⇓
, serum PSA levels were significantly reduced in the mice that were
castrated or treated with finasteride or L-39 (all
P < 0.02), and L-10 (P < 0.05). Serum testosterone levels were significantly reduced in
the groups treated with castration (P < 0.001), L-37 (P < 0.02), and L-39
(P < 0.05). Tumor testosterone was
significantly reduced (P < 0.001) in the
mice treated by castration, and of the novel compounds tested, only
L-37 significantly reduced (P < 0.02) tumor
levels of testosterone.

The effects of orchidectomy, finasteride, ketoconazole,
and the novel compounds L-10, L-37, and L-39 on the growth of LNCaP
prostate tumors in male SCID mice. Groups of six mice with LNCaP tumors
were treated with the compounds at 50 mg/kg/day for 28 days.
A, tumor volumes were measured weekly, and the
percentage of change in tumor volume was determined. B,
after 28 days of treatment, the mice were sacrificed and the tumors
were removed and weighed. Castration and L-39 significantly reduced
tumor weights. ∗, P < 0.05
versus control.

The effects of L-39 on the growth of hormone-dependent PC-82 tumor
xenografts were determined because these tumors express wild-type AR.
Castration, flutamide, and finasteride were used as the reference
treatments. The mice were grouped approximately 21 weeks after tumor
inoculation, when measurable tumor volume was approximately 500
mm3 (Fig. 9A)
⇓
. Tumor volume in the control mice increased 2.2-fold over
the 28 days of treatment, and tumors in the castrated mice actually
regressed by 13% over the duration of the experiment. Compared with
the control mice, finasteride slowed tumor growth but not to the same
extent as the antiandrogen flutamide. Tumor volume in the mice treated
with flutamide increased by only 1.1-fold over the 28 days of
treatment. L-39 was almost as effective as flutamide at slowing the
growth rate of the PC-82 tumors. Tumor volume in the mice treated with
L-39 increased by 1.3-fold during the course of the experiment. These
results were also reflected in the weights of the tumors. Compared with
the control group, castration, flutamide, and L-39 all significantly
lowered (P < 0.02) PC-82 tumor weight (Fig. 9B)
⇓
and serum levels of PSA (Table 4)
⇓
. Castration also significantly lowered (P < 0.01) serum and tumor levels of testosterone. Flutamide had no effect
on the serum levels of testosterone. Serum and tumor testosterone
levels were significantly lower (P < 0.01)
in the mice treat with L-39.

The effects of orchidectomy, finasteride, flutamide, and
the novel compound L-39 on the growth of PC-82 prostate tumor
xenografts in male athymic nude mice. Groups of five mice with PC-82
prostate cancer tumors were treated with the compounds at 50 mg/kg/day
for 28 days. A, tumor volumes were measured weekly, and
the percentage of change in tumor volume was determined.
B, after 28 days of treatment, the mice were sacrificed
and the tumors were removed and weighed. Castration, flutamide, and
L-39 significantly reduced tumor weights. ∗,
P < 0.02 versus
control.

Serum levels of PSA and testosterone and tumor levels of testosterone
in male athymic nude mice with PC-82 tumors after 28 days of treatment
with the novel compound L-39

DISCUSSION

In the present study, we evaluated the ability of novel inhibitors
of either C17,20-lyase and/or 5α-reductase to
inhibit the growth of hormone-dependent prostatic cancer cells in
vitro and in vivo. We had reported previously that
several of these novel compounds showed very good biological activity
by lowering androgen levels in a normal male rat model, suggesting that
they may be effective treatment options for hormone-dependent prostatic
carcinoma
(47, 48)
. In this report we describe that some
of our novel androgen synthesis inhibitors also interact with the AR
expressed in prostate cancer cells, and that the antiandrogenic
properties of some of the compounds contribute, at least in part, to
their ability to inhibit the growth of hormone dependent prostate
cancers.

As an initial approach, the effects of the compounds on the growth of
hormone-dependent LNCaP cells were determined. Of the six novel
compounds tested, three stimulated LNCaP cell growth (L-2, L-10, and
low concentrations of L-36), two inhibited cell proliferation (L-35 and
L-37), and one had no effect (L-39). The mitogenic effects of L-2 and
L-10 were surprising because both of these compounds are pregnane
derivatives and would not expected to be recognized as ligands by the
LNCaP AR. However, the LNCaP AR contains a mutation in the
ligand-binding domain (threonine to alanine at residue 877) that is
responsible for it recognizing other steroidal compounds as either
agonists or antagonists
(33, 34)
. It has recently been
reported that residue 877 contacts the ligand directly, and the
mutation alters the stereochemistry of the binding pocket
(49)
. The mutation broadens the specificity of ligand
recognition and the LNCaP AR recognizes estrogens, progestins, and
antiandrogens such as flutamide as androgens. The cells respond to
these compounds with increased proliferation
(34, 50,
51,
52)
.
Consistent with previously published reports from this laboratory,
flutamide was found to be growth stimulatory to LNCaP cells and
finasteride was growth inhibitory (Fig. 2
⇓
; Refs.
45 and 46
). Our results show that the effects of flutamide and
finasteride are specific for the LNCaP AR and not for the wild-type AR.
This is in contrast to the results obtained with our novel compounds,
which interacted with both AR types and maintained the same
agonistic/antagonistic properties regardless of receptor type. This
indicates that some of our antiandrogenic compounds may be useful for
the treatment of patients with tumors expressing either wild-type or
mutated AR, or for patients with amplified AR expression. Androgen
responsive cells are believed to respond to androgen ablation therapy
by acquiring androgen-independence and adapting to growth in the
absence mitogenic androgens. However, recent studies have suggested
that in a subset of tumors, cells respond to reduced androgen levels by
amplifying AR gene expression
(53, 54)
. Therefore,
AR-mediated signaling is likely to be important in the advanced-stage
disease. Compounds such as L-39, which is a potent inhibitor of
C17,20-lyase and 5α-reductase and exhibits
antiandrogenic properties, may be a suitable treatment option for such
patients.

Prostate cancer cells with mutated AR respond to flutamide and
hydroxyflutamide, the active metabolite of flutamide, with growth
stimulation rather than growth inhibition. Our studies, in
vitro and in vivo, used flutamide, because we have
shown previously that LNCaP cells respond equally well to both
compounds
(45, 46)
. Findings of growth stimulation by
flutamide led to the development of bicalutamide (Casodex), a
second-generation, nonsteroidal antiandrogen
(56, 57)
.
Bicalutamide has a 4-fold-higher affinity for the AR than flutamide and
is recognized by the LNCaP AR as an antiandrogen. Clinically,
bicalutamide is providing better responses and is better tolerated than
flutamide
(57)
. Experiments ongoing in this laboratory are
presently comparing the antitumor effects of bicalutamide to some of
our more potent novel compounds. Although the LNCaP prostate cancer
cell line is the most characterized androgen-dependent model of
prostate cancer, the mutation in the AR is problematic for
investigators researching the effects of antiandrogens. Furthermore,
LNCaP cells also respond to androgens in a biphasic
concentration-dependent response, with high concentrations of androgens
inhibiting cell proliferation
(58)
. We observed this with
L-36, which was a potent stimulator of cell proliferation at the lower
concentrations and a potent inhibitor of cell growth at the
5-μm concentration. Therefore, whereas the
competitive binding studies with the synthetic androgen[
3H]R1881 indicate that the compounds
are interacting with both the mutated LNCaP AR and the wild-type AR,
they cannot discriminate whether the compounds are acting as agonists
or antagonists. We used transcriptional activation assays with
luciferase activity regulated by an androgen responsive promoter to
overcome these difficulties. LNCaP-LUC cells were selected in this
laboratory and responded to DHT and testosterone in a manner similar to
the parent cells, indicating that no clonal variation had occurred
during the selection process. The studies confirmed that the growth
stimulatory compounds (L-2, L-10, and L-36) were functioning as pure AR
antagonists whereas the growth inhibitory compounds (L-35, L-37, and
L-39) were AR antagonists. It is interesting to note that the pregnane
derivatives L-2 and L-10 were agonists of both receptor types. This
suggests that the side chain modification at carbon 17 may play an
important role in mediating these effects. L-35, L-37, and L-39 were
also determined to be antagonists of both the wild-type AR and the
LNCaP AR, indicating that they are more comparable with the
second-generation antiandrogen bicalutamide than with flutamide.

Having established the in vitro activities of the novel
compounds, the in vivo effects were determined using LNCaP
prostatic cancer cells grown as tumor xenografts in male SCID mice. In
a preliminary experiment (data not shown), the growth of LNCaP prostate
cancer xenografts in male SCID mice was determined to be
hormone-dependent, and tumor weights were significantly lower in the
animals that were castrated or treated with the
5α-reductase-inhibitor finasteride. Flutamide was found to stimulate
the growth of LNCaP tumor xenografts, but not to the same extent as
DHT. Additionally, the maximum time of treatment was determined to be
28 days. After this time, tumors in the castrated group appeared to
have acquired hormone-independence based on a determination of weekly
serum PSA levels (data not shown).

In agreement with the results obtained in vitro, the
androgenic compounds L-2, L-36, and L-10 had no appreciable effect on
tumor growth measurements. L-10 was found to be as effective as
finasteride at inhibiting tumor growth, and L-2 and L-36 significantly
lowered serum levels of testosterone but not tumor testosterone levels.
Therefore, it is likely that although the compounds are effective
inhibitors of androgen synthesis, this is overshadowed by their
androgenic properties, which results in LNCaP tumor growth. The
inability of L-35 and L-37 to inhibit LNCaP tumor growth in
vivo was especially disappointing. Both of these compounds were
the most effective at inhibiting LNCaP cell growth in vitro,
and they were both determined to be the most potent antiandrogens. We
have recently determined that L-37 is an inhibitor of the liver
cytochrome P450 enzymes 3α-hydroxysteroid-oxidoreductase and
3β-hydroxysteroid-oxidoreductase, which catalyze the metabolism of
DHT to polar metabolites that are excreted in the urine (data not
shown). Although we were unable to reliably measure serum levels of DHT
in the serum of the animals because of cross-reactivity with
testosterone and the novel compounds, the results (not shown) did
suggest that there were elevated serum DHT levels in the animals
treated with L-37. These elevated DHT levels may explain why L-37 had
no effect on tumor growth in vivo. It should also be noted
that L-37 does not inhibit the growth of PC-82 prostate cancer
xenografts grown in athymic nude mice in vivo (data not
shown). Currently, we do not know the reasons for the lack of growth
inhibition observed with L-35. The inability of L-35 to inhibit LNCaP
tumor growth may be attributed to its weak affinity for the AR.
Alternatively, it may be attributable to the compound being converted
to an inactive metabolite, poor uptake of the compound in the animals,
or rapid clearance from the animals.

The most effective compound at inhibiting LNCaP tumor growth in
vivo was L-39, which paralleled castration. Moreover, L-39 was
also very effective at inhibiting the growth of hormone-dependent PC-82
prostate tumor xenografts in athymic nude mice. PC-82 prostate cancer
cells expresses wild-type AR, and the cells cannot be grown in
vitro. In these experiments, PC-82 tumors grew to a measurable
size in approximately 12 weeks. The in vitro experiments had
determined that L-39 was effective at inhibiting LNCaP cell growth in
the presence of exogenous steroids, and that L-39 is extremely
effective at preventing high affinity androgens from binding to the AR.
However, when compared with L-35 and L-37, L-39 was not the most potent
antiandrogen. We have recently reported on the pharmacokinetic
profile of L-39 in normal (non-tumor-bearing) male mice
(59)
. L-39 (50 mg/kg s.c.) was shown to be cleared from
the blood of the animals within 6 h of injection, and a nonpolar
metabolite was detected in the serum. The metabolite has not yet been
characterized, and it is not yet known whether it is contributing to
the antitumor activities of L-39. It is also important to note that the
multifunctional activities L-39 are most likely to explain the
antitumor effects of this compound. L-39 is an inhibitor of androgen
synthesis, and it is a potent antiandrogen when tested against both the
mutated LNCaP AR and the wild-type AR. Although additional studies are
required to determine the full potential of this compound, L-39 holds
considerable promise as a treatment option for hormone dependent
prostate cancer.

Acknowledgments

We are grateful to Dr. John Isaacs (The John Hopkins School of
Medicine, Baltimore, MD) for providing the doner PC-82 tumor
xenografts, to Dr. Marco Marcelli (Baylor College of Medicine, Houston,
TX) for providing PC-3 cells and PC-3 cells stably transfected with the
human wild-type androgen receptor, to Dr. Elizabeth Wilson (University
of North Carolina, Chapel Hill, NC) for providing the wild-type
human AR-expressing vector and the LNCaP AR-expressing vector, to Dr.
Hynda Kleinman (NIH, Bethesda, MD) for providing the Matrigel, and to
Merck Research Laboratories (Rahway, NJ) for providing the finasteride.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by NIH Grant CA-27440 and a grant from
Paramount Capital, Inc., New York, New York.

Sarosdy M. F. Which is the optimal antiandrogen to use in combined androgen blockade of advanced prostate cancer?.
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